The present invention relates to methods of increasing biomass in plants and plants generated thereby.
Plants specifically improved for agriculture, horticulture, biomass conversion, and other industries (e.g. paper industry, plants as production factories for proteins or other compounds) can be obtained using molecular technologies.
Availability and maintenance of a reproducible stream of food and animal feed to feed animals and people has been a high priority throughout the history of human civilization and lies at the origin of agriculture. Specialists and researchers in the fields of agronomy science, agriculture, crop science, horticulture, and forest science are even today constantly striving to find and produce plants with an increased growth potential to feed an increasing world population and to guarantee a supply of reproducible raw materials. The robust level of research in these fields of science indicates the level of importance leaders in every geographic environment and climate around the world place on providing sustainable sources of food, feed, chemicals and energy for the population.
Manipulation of crop performance has been accomplished conventionally for centuries through plant breeding. The breeding process is, however, both time-consuming and labor-intensive. Furthermore, appropriate breeding programs must be specially designed for each relevant plant species.
On the other hand, great progress has been made in using molecular genetic approaches to manipulate plants to provide better crops. Through introduction and expression of recombinant nucleic acid molecules in plants, researchers are now poised to provide the community with plant species tailored to grow more efficiently and produce more product despite unique geographic and/or climatic environments. These new approaches have the additional advantage of not being limited to one plant species, but instead being applicable to multiple different plant species (Zhang et al. (2004) Plant Physiol. 135:615).
Despite this progress, today there continues to be a great need for generally applicable processes that improve forest or agricultural plant growth to suit particular needs depending on specific environmental conditions.
Cellulose, the most abundant organic polymer in the world, is deposited in the stems of plants and is extensively utilized for fuel, timber, forage, fibre and chemical cellulose.
Cellulose synthesis, in contrast with starch, is essentially an irreversible sink. Cellulose is produced from the precursor UDP-glucose, which can be formed via two potential pathways. UDP-glucose can be derived from the cleavage of sucrose in a reaction catalyzed by sucrose synthase (SuSy; EC 2.4.1.13) yielding UDP-glucose and fructose. Alternatively, UDP-glucose can be generated from the phosphorylation of glucose-1-phosphate in a reaction catalyzed by UDP-glucose pyrophosphorylase (UGPase, EC 2.7.7.9).
Another potential source of UDP-glucose is galactose. The entry of free galactose into metabolism begins with its phosphorylation by galactokinase (EC 2.7.1.6) to Gal-1-P. Following phosphorylation, two alternative pathways exist for the fate of the Gal-1-P in plants. One pathway is via the Leloir reaction, carried out by a uridyltransferase (UT, UDP-Glc: Hexose-1-P uridyltransferase, EC 2.7.7.12) utilizing UDP-Glc in a transferase reaction. However, this enzyme is generally not observed in most plants.
In an alternative pathway, Gal-1-P may be converted into UDP-Gal via a pyrophosphorylase (PPase, Gal-1-P: UTP transferase) utilizing UTP:Gal-1-P+UTP←→PPi+UDP-Gal. 
The UDP-Gal product of this pathway is further metabolized to UDP-Glc via the epimerase reaction.
Previous studies have shown that the melon fruit, with its active Gal metabolism, shows little UT activity, suggesting that a PPase is responsible for Gal-1-P metabolism [Smart and Pharr, 1981, Planta 153: 370-375; Feusi et al., 1999, Physiol Plant 106: 9-16]. There is no known PPase that is specific for the Gal moiety in melon fruit [Smart and Pharr, 1981, Planta 153: 370-375; Feusi et al., 1999, Physiol Plant 106: 9-16]. Rather, there appears to be a PPase in melon fruit which can utilize both Gal-1-P and Glc-1-P. This dual substrate PPase is present in cucurbit fruit in addition to the UGPase (UDP-Glc PPase, E.C. 2.7.7.9) which is specific for the Glc-1-P sugar, and inactive with Gal-1-P [Smart and Pharr, 1981, Planta 153: 370-375; Feusi et al., 1999, Physiol Plant 106: 9-16; Gao et al., 1999, Physiol Plant 106: 1-8]. Feusi et al. (1999) purified and characterized an enzyme fraction from melon fruit which catalyzed the nucleotide transfer to both Glc-1-P and Gal-1-P and were unable to further separate the activities, suggesting that the two reactions are catalyzed by the same protein (a UGGPase).
A UGGPase enzyme was described in germinating pea seeds (Kotake et al., 2004, J Biol Chem. 2004 October 29;279(44):45728-36). The enzyme catalyzed the formation of UDP-Glc, UDP-Gal, UDP-glucuronic acid, UDP-1-arabinose, and UDP-xylose from respective monosaccharide 1-phosphates in the presence of UTP as a co-substrate, indicating that the enzyme has broad substrate specificity toward monosaccharide 1-phosphates.
It has been shown that there is a correlation between plant cellulose content and overall biomass. For example, a gene for UDP-glucose pyrophosphorylase has been cloned, and sense constructs inserted in tobacco plants. Heightened enzyme activity and cellulose synthesis were reported [Xue et al. 1997, Plant Physiol. 114(suppl 3):300]. Analyses indicated a 30% enhancement of cellulose content and a 20% increase in biomass. In addition, Coleman et al [Plant Biotechnology Journal. 4: 87-101, 2006] teach transgenic expression of UDP-Glc PPase in aspen trees and show a significant increase in plant height and biomass.
There is thus a widely recognized need for, and it would be highly advantageous to identify novel enzymes which utilize both glucose and galactose substrates for increasing cellulose content and biomass in plants.